Processing and Function of the Gp2/3/4 Spike of the Porcine Reproductive and Respiratory Syndrome Virus (PRRSV)

• Applicant: Privatdozent Dr. Michael Veit
• Subject Area: Veterinary Medical Science
• Term: from 2014 to 2020
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 256219832

Arteriviridae are a family of enveloped RNA viruses, which are, despite their importance in veterinary medicine, only poorly characterized. The equine arteritis virus (EAV) is the prototype member of the family and causes substantial disease in horses, whereas the porcine reproductive and respiratory syndrome virus (PRRSV) is the most important pathogen in the porcine industry. PRRSV causes persistent infection, which is, besides the high variability of its glycoproteins, the main obstacle to eliminate the virus from pig farms. One possible molecular cause of persistence is the late appearance of neutralizing antibodies, but their protein targets and epitopes have not been identified. The envelope of arteriviruses contain two glycoprotein spikes, Gp5/M and Gp2/3/4, which are in cells retained in the ER or early-Golgi, the assembly site of virus particles. Reverse genetic experiments indicate that Gp5/M is required for budding and Gp2/3/4 for cell entry. Gp2/3/4 is supposed to attach arteriviruses to cellular receptors and to catalyse membrane fusion. The disulphide-linked Gp2/3/4 spike is assembled by a complicated and largely uncomprehended process. Whereas Gp2 and Gp4 form a disulphide-linked dimer inside cells, presumably in the ER, disulphide-linkages between Gp3 and Gp2/4 only form in budded virus particles. Our recent work with EAV revealed another unique feature of Gp3: N-linked carbohydrates located adjacent to the signal peptide inhibit its cleavage. These studies also led to a new model for the membrane topology of Gp3: The uncleaved signal peptide does not act as a membrane anchor but is completely translocated into the lumen of the ER. Anchoring is caused by the hydrophobic C-terminus, which is not a transmembrane region, but attaches Gp3 peripherally to membranes. With our research project we want to unravel the assembly pathway of Gp2/3/4 and create a tool to analyse its function. We shall first analyse whether Gp3 from various PRRSV strains follows the same unique processing scheme and exhibits the same membrane topology as Gp3 from EAV. Interestingly, PRRSV strains differ in the biophysical properties of the C-terminus of Gp3 suggesting that in some strains Gp3 might not be membrane bound, but secreted from cells. Reverse genetics will be used to analyse the significance of membrane anchoring of Gp3 on the infectivity of viruses. We shall then assemble a native Gp2/3/4 complex in transfected cells from its components. Removal of retention signals, which have not been identified but are likely to be located in the transmembrane region of the proteins, will allow targeting of the complex to the plasma membrane. A surface exposed Gp2/3/4 complex is amenable to a multitude of examination methods, such as attachment to cellular receptors, binding to antibodies and membrane fusion assays and is thus instrumental to elucidate the role of the Gp2/3/4 spike during virus entry and as a putative target for neutralizing antibodies.

• DFG Programme: Research Grants